Phorboxazoles A and B are structurally complex natural products isolated from Indian Ocean sponges. These natural products display extraordinarily potent cytostatic activity towards the US National Cancer Institute's panel of 60 human cancer lines (mean GI50 values of <1.6 x 10-9 M;most cell lines were 100% inhibited at this lowest test concentration) and represent an entirely new class of cytostatic agents, having been characterized as being "among the most potent cytostatic agents yet discovered." The phorboxazoles'mechanisms of action involve S phase cell cycle arrest and induction of apoptosis. Total synthesis has provided small amounts of the natural products and structural analogs that have been used to identify the importance of several structural features for potent activity and new lead compounds. Fluorescent derivatives of phorboxazole A have been developed and used to isolate phorboxazole analog binding proteins from HeLa cells. Phorboxazole analogs were independently found to bind to cyclin dependent kinase 4 (cdk4), cytokeratins, and several other proteins. The sequestration of cdk4 on cytokeratin intermediate filaments induced by phorboxazole analogs may cause S phase cell cycle arrest. However, the biological effects of phorboxazole interactions other targets needs to be fully elucidated. The overall goal of this proposal is to continue to develop the phorboxazole chemotype as a promising new class of anticancer therapeutic agents by innovating further synthetic organic and biological chemistry to uniquely and rapidly access structural analogs of the phorboxazoles, validate their cellular targets and define the essential biomolecular interactions, determine the essential phorboxazole pharmacophores, and provide mmole quantities of active analogs. A novel and rapid tricomponent approach to access the phorboxazole molecular architecture has been developed and will be coupled human cancer cell cytotoxicity and selectivity screens to map the natural product's essential pharmacophores and identify simpler molecules with favorable activity profiles. An array of biophysical studies will be used to detail ligand-receptor interactions and assist in target validation. The successful outcome of this project will completely define the mode and mechanisms of phorboxazoles'cytostatic and apoptotic activities, and will provide unique small molecule therapeutic candidates. This project seeks to identify and exploit novel cellular targets involved in the progression of the cell cycle of cancer cells using synthetic analogs of the phorboxazole natural products.